PROJECT SUMMARY Acute rejection (AR) has a major impact on the survival of liver transplant (LT) recipients. The strategy that providers currently use is to slowly reduce immunosuppressive (IS) therapy over time, using blood trough levels to guide weaning. However, these levels do not correlate well with the degree of IS required, and as such, nearly 20% of patients experience AR during tapering that can lead to complications from graft injury or from the adverse effects of IS therapy. These approaches might be imminently improved if proactive strategies to detect AR non-invasively before it is clinically established were available. Our group has pioneered a novel biomarker platform called ?top down? proteomics (TDP), which measures intact modified proteins (`proteoforms') in peripheral blood mononuclear cells (PBMC) which may serve as high quality biomarkers to detect early immune activation seen in AR. This proposal aims to determine whether novel PBMC proteoforms can predict and diagnose AR so as to design future biomarker-guided interventional studies to minimize AR in LT recipients. Our long-term goal is thus to utilize proteomic biomarkers to identify AR in LT recipients and, hence, suitable candidates for interventions aimed at reducing this risk. As a step in that direction, the objective of this R21 application is to validate the accuracy of specific PBMC proteoforms in predicting and diagnosing AR following LT. We hypothesize that that proteoforms will be predictive of AR and distinct from those seen in non-AR graft injury or normal function. The study is planned with the following three specific aims: Aim 1. To confirm the accuracy of specific proteoforms in diagnosing acute rejection following liver transplantation. Aim 2. To test the ability of proteoforms to predict acute rejection following liver transplantation. Aim 3. To enhance the understanding of mechanistic pathways and prediction of rejection by correlating validated genomic and proteomic expression signals. We are uniquely positioned to conduct this research because: 1) Viable PBMC samples [Northwestern; NIAID CTOT14 trial (U01 AI084146)] collected from LT recipients before and at the time of AR, as well as other phenotypes as controls, are readily available to analyze for proteoform signatures; 2) A unique, established collaboration exists within Northwestern between the Comprehensive Transplant Center that houses the biorepository and the Proteomics Center of Excellence which have already acquired preliminary discovery data on proteoforms of AR in our organ transplant recipients. The approach will be to perform internal and external validation of diagnostic proteoform signatures of AR in independent cohorts (NU & CTOT14 patient samples). We will then test serial CTOT14 samples to evaluate the evolution of protein signatures prior to AR, and correlate these results with gene expression data, providing mechanistic insights into graft injury and refining prediction. Upon completion, we expect to have identified specific biomarkers that can predict and diagnose AR with enough accuracy for use in future interventional studies aimed at reducing AR and its complications. 1